Drug resistance is a significant problem in cancer therapy, because it is a general phenomenon among all malignancies and there is no effective solution. Therefore there is an unmet clinical need for new therapies to be developed targeting drug-resistant malignancies, which is challenging due to the limited knowledge about drug resistance mechanisms and the lack of appropriate cancer models and research tools. Our long-term goal is to elucidate the mechanisms whereby cancer cells acquire resistance to treatment, and to rationally develop antitumor agents based on such knowledge that will effectively treat drug-resistant malignancies. Drug-resistant leukemias over-express anti-apoptotic Bcl-2 family proteins and p-glycoprotein. Some also have elevated levels of Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA), which has been reported to interact with the Bcl-2 protein. We have recently identified a set of small molecules, derived from HA 14-1 (a putative Bcl-2 inhibitor) and termed CXLs, that demonstrate preferential anticancer activity toward drug- resistant leukemias, despite the fact that such cancers reveal cross resistance to standard therapies. Leukemia cells also fail to develop resistance to CXLs. In fact, CXLs re-sensitize leukemias to standard therapies (30-130 fold). Mechanistically, CXLs induce ER Ca2+ release and ER stress, inhibit SERCA and p- glycoprotein. These data overall demonstrate the unique biological features of CXLs, particularly their potential to treat drug-resistant leukemias. Nevertheless, the precise mechanism that accounts for the distinctive anticancer profile of CXLs, including CXL's cellular targets, has yet to be firmly established. During the course of our preliminary studies, we have developed two structurally similar CXLs as chemical probes and several pairs of leukemia cell lines as cancer models for mechanistic investigation. The main objectives of this proposal are to elucidate the mechanisms of action for CXLs by employing the unique chemical probes and cancer models we developed and to validate its anticancer potential in a clinically relevant engraftment model. The resulting knowledge is expected to rationalize the selective anticancer activity of CXLs towards drug- resistant leukemias and to establish the foundation for future translational development. Aim 1. To define the contribution of the anti-apoptotic Bcl-2 family proteins & SERCA proteins to drug resistance and their roles in CXL's selective anticancer activity. Aim 2. To profile the proteins that CXL positive lead preferentially interacts with in drug-resistant cancer cells, which we hypothesize to be the cellular targets for CXLs and be responsible for drug resistance. Aim 3. To validate the anticancer selectivity and mechanism of action for CXL positive lead against drug resistant AML in engraftment models.